Germline mutations in mismatch repair genes are the most common cause of hereditary colon cancer. Mutations in four mismatch repair genes (MSH2, MLH1, PMS1 and PMS2) have been identified primarily in families with hereditary nonpolyposis colorectal cancer (HNPCC) featuring high penetrance, early age at diagnosis, and right colon predominance. These patients tend to show replication errors (microsatellite instability (MSI) in DNA repeat sequences of their neoplastic cells. Recently, inherited mutations in a fifth mismatch repair gene, MSH6, have been identified in colorectal cancer patients. Concurrently, our preliminary studies have found a total of 9 germline MSH6 mutations in 198 patients with colorectal cancer and diverse family histories. Surprisingly, none of our MSH6 carriers have family histories that fulfill the classic Amsterdam criteria for HNPCC; instead, they generally have only one first- or second degree relative with cancer. In addition, the MSH6 carriers have a median age of diagnosis of colorectal cancer of 62.5 years, similar to that of sporadic cases. Family members are affected by a variety of other cancers, including breast, endometrial and ovarian tumors. To pursue these unexpected observations, we propose to analyze the MSH6 gene in 700 CRC cases with family histories that do no fulfill classic criteria for HNPCC. Cases will be stratified by age at diagnosis. Cases with germline MSH6 mutations and an equal number of controls of colorectal cancer cases without MSH6 mutations will be analyzed for MSI. Additionally, family members of MSH6 carriers will be evaluated for the germline mutation, and their available tumor blocks will be examined for MSI. Results will be analyzed to determine 1. the frequency and clinical manifestations of germline MSH6 mutations in all 700 colorectal cancer patients; 2. types and frequencies of MSI in the corresponding tumor tissues of MSH6 carriers as compared with a matched series of non-carriers; and 3. MSH6 status of affected and unaffected relatives of MSH6 carriers, as well as MSI in tumors of affected family members. Our preliminary data suggest that MSH6 may be responsible for more colorectal cancer cases than MSH2, MLH1, PMS1 and PMS2 combined. Evidence that MSH6 is a moderately penetrant, later-onset hereditary colorectal cancer gene would imply the existence of similar genes for other common cancers that await discovery.